C. Bertoni-Freddari, P. Fattoretti, T. Casoli, G. Di Stefano, B. Giorgetti, Y. Grossi, M. Balietti

A growing body of experimental results is supporting the critical role played by mitochondrial damage in physiological aging and age-related pathologies. This clear awareness has prompted the need of checking the functional state of mitochondria in order to plan adequate intervention strategies to counteract mitochondrial dysfunctions. In this context, the activity of cytochrome oxidase (COX) is presently considered as a reliable marker of neuronal metabolism. COX, an integral transmembrane protein of the inner mitochondrial membrane, is the terminal enzyme of the electron transport chain and its activity can be preferentially evidenced within the mitochondria, at the sites where the enzyme is located, by the diaminobenzidine cytochemical technique. Computer-assisted morphometric studies have been carried out on COX-positive mitochondria in the nervous system of adult and old rats to assess the metabolic competence (MMC) of synaptic organelles in aging. Numeric density (Nv), volume density (Vv), average mitochondrial volume (V) and length (Fmax) as well as R (the ratio between the overall area of the cytochemical precipitate due to COX activity and the area of the mitochondrion) were the parameters measured at the distal dendrites of the hippocampal dentate gyrus. In the cerebellar cortex, R was estimated on mitochondria of different size. No significant difference due to age was found as regards Nv, Vv, V and Fmax while R was significantly decreased by 9.3% in the dentate gyrus of old animals. In the cerebellar cortex, the value of R significantly decreases in small and medium-sized organelles by 31.6% and 21.4%, respectively, while in oversized mitochondria (average area: 0.2 mm2) it is almost the same in adult and old rats (0.30 vs. 0.29, respectively). It is well known that mitochondria are in a very plastic condition and that this is responsible of consistent changes of their ultrastructural features: actual energy demands are reported to be able to modulate size, numeric density and volume fraction of the organelles present in discrete cellular compartments. Although no functional significance has been associated to any rearrangement in mitochondrial morphology, the presence of enlarged organelles (also referred to as megamitochondria) within a cell have been supported to represent a sign of adverse conditions sometimes threatening cell survival. On the basis of these concepts, the present findings support that the higher values of Vv and V found in the hippocampus of old rats may be interpreted as compensating reactions masking subtle functional alterations, while the 5.5% increase of Fmax vs. the 9.3% decrease of R suggest that mitochondrial elongation correlates inversely with MMC. This last assumption finds a well grounded support in the present results on COX-positive organelles from the cerebellar cortex. Previous studies from our laboratory showed that the percent of small and medium-sized mitochondria significantly decreases in the cerebellar cortex of the old rats, thus, since this fraction accounts for 80-95% in a given mitochondrial populations, it plays a major critical role in the age-related decay of MMC. By matching quantitative cytochemical measurements of COX activity within single mitochondria with the morphometric assessment of their ultrastructural features, the present results suggest that the estimation of the R value may serve as a reliable mitochondrial morphofunctional check up.

Keywords (Optional): 
Mitochondrial metabolic competence
Cytochrome oxidase
COX cytochemistry
Mitochondrial morphometry